Dengue is transmitted mainly by the Aedes aegypti mosquitoes which inhabit the tropics, making Dengue endemic to these areas. It is caused by four genetically and serologically related viruses, termed DENV1, DENV2, DENV3, and DENV4. Dengue infection is a leading cause of illness and death in the tropics and subtropics, including Puerto Rico and the U.S. Virgin Islands, where thousands of U.S. citizens develop dengue fever every year. With three billion of the world's population at risk from dengue, an estimated 50-100 million cases of Dengue Fever (DF), and hundreds of thousands of cases of severe dengue (previously known as Dengue Hemorrhagic Fever or DHF) that occur every year, the demand for a rapid, sensitive, and serotype- specific dengue diagnostics test is high. Patients would be diagnosed sooner to receive treatment, and public health laboratories would have a clearer picture of the true number of dengue cases. Dengue is an acute illness where most patients are present with symptoms when they are viremic, but have not yet developed antibodies. Therefore, the early detection of viral components such as RNA or antigens is critical. Real-time reverse transcription polymerase chain reaction (RT-PCR) assays targeting the dengue virus in whole blood or serum have been demonstrated to provide serotype-specific and early diagnostics, but it also requires sophisticated instruments to perform the test. In this SBIR Phase I application, we propose to develop a highly specific and sensitive system to detect the presence of dengue viral RNA. Using our self-contained sample preparation cartridge (SPC) technology and a highly robust convective PCR thermal cycler, we will build a low- cost, multi-sample, and integrated system that takes full advantage of a CDC developed, FDA-approved serotype specific real-time RT-PCR dengue diagnostic assay. Nucleic acid extraction in low-resource settings (LRS) for molecular diagnostics is difficult to perform, but through the use of our self-contained SPC, NA extraction can be done in a safe and enclosed environment in less than 10 min. The extracted NA (RNA in this case) can be ejected from the SPC and used for real-time RT-PCR reaction using a rapid and robust convective PCR thermal cycler. Convective PCR is significantly simpler to operate than traditional PCR while maintaining the specificity, sensitivity, and multiplex capability of PCR. The speed of cPCR is also comparable to isothermal amplification. Using our system, we will enable identification of the serotype and quantitative determination of the viral load in less than 30 min of RT-PCR reaction. Unlike other single-sample integrated systems that are being developed, our approach allows multi-sample processing and molecular detection without significantly adding cost to the system. This means our approach will lead to an assay that can be practically and cost-effectively implemented in LRS. We will work closely with the CDC Dengue Branch in Puerto Rico to integrate the serotype-specific dengue RT-PCR assay into our system to deliver an effective dengue assay without compromised performance. At the conclusion of the Phase I, we will demonstrate the technology to our collaborator at CDC's Dengue Branch.